The present invention relates generally to the electrophoretic separation of biomolecules, including but not limited to, proteins, peptides, DNA and RNA.
Two-dimensional gel electrophoresis is well known. Using this technique, a complex mixture of biological material can be resolved into its components based on two independent variables, such as charge and mass. A first gel separation, based on the isoelectric point of biomolecules as one method of separation, is generally run in a tube. The biological sample is placed at the top of a tube containing a gel and an electric current is applied. After electrophoretic separation of the biomolecules, the gel is then removed from the tube and placed across the top of a slab gel and the second method of separation is performed. The resulting two-dimensional array will display discrete spots that are not separable with any single electrophoretic separation.
Many methods of electrophoretic separation or sample treatment are known and have been used in various combinations for two-dimensional gel electrophoresis. These include polyacrylamide gel electrophoresis (PAGE: separation by charge-mass migration in an electric field), SDS-PAGE (separation by mass of sample treated with detergent), isoelectric focusing (IEF: separation by isoelectric point in a pH gradient), and enzymatic digestion after the first separation and before the second separation. All of these methods are well known to those skilled in the art of analysis of biological materials and the choice such an artisan will make is dependent on the nature of the material and the information sought to be elucidated.
For purposes of electrophoresis, it is necessary to form the gel by polymerization of monomers in a support. The properties of the gel can be varied as desired by varying the monomer content and the degree of cross-linking. The resulting gels are fragile, particularly those of low polymer content, such as five percent (5%), which are useful for separating molecules of high molecular weight. The most commonly used support has been glass. However, glass has a strong surface negative charge causing electroosmotic flow, which distorts the separation. Removal of the gel from the first run can be difficult because of the attraction of the gel to the glass. In addition, high frictional coefficients add to the problem of removal, particularly when very small-bore tubing, that is, capillaries, are used.
U.S. Pat. No. 5,447,617 describes covalently bonding polybutadiene to the inner surface of a capillary tube in an attempt to eliminate these problems. U.S. Pat. No. 4,680,201 reaches the same result by bonding a monomer layer to the inner surface of some glass tubes and polymerizing the monomer in situ. U.S. Pat. No. 5,858,188 discloses microchannels in the form of a variety of configurations, including capillaries, having an inner surface coated with acrylic polymer.
The need remains to provide an improved gel support for electrophoresis.
This invention comprises an improved gel support tube formed of a selectively permeable microporous polymer for use in electrophoresis. The microporous tube is selected to be non-wettable during the first procedure, preventing the sample from escaping through the micropores. Such microporous tubes have been made, by way of example and without limitation, from polyethylene, polypropylene, polystyrene, polycarbonate, polysulfone and the like. The sizes of the pores are easily varied. The pore size chosen is dependent on the substances to be separated, with larger pores being more useful for large molecules. The only limitation on material and pore size is that the material must be nonwettable in the absence of detergent or organic solvent and wettable in the presence of detergent or organic solvent. The pore size must be large enough so as not to impede the passage of large molecules nor so small that biological samples leak through during the first separation. Following electrophoretic separation of biological materials the separated components can be removed through the pores of the support by treatment with detergent, which renders the pores permeable to the sample, followed by electrophoresis or absorption onto a surface.
The size of the support tube can be varied according to whether the separation is preparative or for the purposes of identification of the separated biological material. Frequently, it is desired to use small samples of material. In that case, the support tube selected will be small and, for purposes of describing this invention, is termed a xe2x80x9cfiber.xe2x80x9d
This invention further comprises a method of performing two-dimensional gel electrophoresis which eliminates the step of removal of the gel from the tube of the first dimensional run before placing the gel in the second dimensional apparatus. The entire tube is placed on the second dimension gel. During the second run, the electrophoretic solution contains a wetting agent, so that the components of the sample may be driven through the pores by the electric field into the second dimension slab gel. The second dimension can be run directly with the gel of the first dimension, or after the gel of the first dimension is subjected to further treatment. A common further treatment is the digestion of the biological material by a degradative enzyme. When the sample is a protein, it is often desirable to break the protein into fragments with a proteinase such as trypsin or papain.
This invention also comprises a means to form an acrylamide gel within a protective sheath so as to prevent ambient oxygen from interfering with polymerization. The support tube is inserted into a polymer sheath, as long as or longer than the support tube and of a diameter just sufficient to allow insertion of the support tube into the protective sheath.
This invention also comprises a means of eluting separated components of a biological sample onto nylon mesh or filter paper.